2007 Fiscal Year Final Research Report Summary
Structural and functional analysis of the motor protein prestin using purified protein
| Project/Area Number |
18390455
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Otorhinolaryngology
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| Research Institution | Tohoku University |
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Principal Investigator |
WADA Hiroshi Tohoku University, Graduate School of Engineering, Professor (30111264)
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| Co-Investigator(Kenkyū-buntansha) |
KOBAYASHI Toshimitsu Graduate School of Medicine, 大学院・医学系研究科, professor (80133958)
KUMAGAI Izumi Graduate School of Engineering, 大学院・工学研究科, Professor (10161689)
IKEDA Katsuhisa Department of Otorhinolaryngology, Juntendo University School of Medicine, Professor (70159614)
TSUMOTO Kouhei Department of Medical Genome Sciences, The University of Tokyo, Associate Professor (90271866)
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| Project Period (FY) |
2006 – 2007
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| Keywords | Otology / Protein / Molecular motor / Prestin / Atomic force microscopy / Purification / Isothermal titration calorimetry |
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| Research Abstract |
Outer hair cells (OHCs), which are sensory cells in the mammalian cochlea, are able to elongate and contract in length in response to changes in memebrane potential. This motility is believed to amplify the motion of the cochlear partition generated by acoustical stimulation. As a result, mammalian hearing is characterized by high sensitivity, wide dynamic range and sharp frequency selectivity. The origin of this motility is associated with conformational change of the motor protein prestin expressed in the lateral wall of OHCs. In the present study, an attempt was made to elucidate the molecular structure and molecular mechanisms of the conformational changes of prestin. First, the plasma membranes of prestin-transfected Chinese hamster ovary (CHO) cells and those of untransfected CHO cells were observed by atomic force microscopy (AFM). More particle-like structures with a diameter of 8-18 nm were found to exist in the plasma membrane of the prestin-transfected CHO cells than in that of the untransfected CHO cells, indicating that the diameter of prestin is 8-12 nm. Second, prestin molecules expressed in the plasma membranes of prestin-transfected CHO cells were labeled with Quantum dots (Qdots), and those dots, about 8 nm in height, were clearly imaged by AFM. Squarish ring-like structures, each with four peaks and one valley at its center, were observed in the vicinity of the Qdots, suggesting that prestin forms a tetramer in the plasma membranes of the prestin-transfected CHO cells. Finally, prestin molecules were purified from prestin-transfected CHO cells. To reveal whether the purified prestin was functional, based on a finding that prestin binds anions, the interaction between purified prestin and chloride was examined by isothermal titration calorimetry. As a result, heat absorption resulting from prestin-chloride interaction was detected, suggesting that the obtained purified prestin was functional.
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